1. Field of the Invention
Certain embodiments of the invention disclosed herein relates to vectors and methods for expressing polypeptide multimers in eukaryotic cells, both in vitro and in vivo, using alternative splicing. Methods for producing cells containing these vectors are included, as well as the use of these vectors and the polypeptides expressed therefrom for the treatment of disease and for the efficient in vivo or in vitro production of such multimeric proteins.
2. Description of Background and Related Art
Polypeptide multimers are assemblies of two or more polypeptides that together form a complex. The polypeptides that make up the complex are usually different. Antibodies are a typical polypeptide multimer in that they are comprised of two antibody light chain polypeptides and two antibody heavy chain polypeptides which together form a tetrameric complex.
The expression of polypeptide multimers in host cells is a challenging process in that the expression of each different polypeptide that makes up the polypeptide multimer must be carefully coordinated. For example, to express an antibody in eukaryotic cells, a first gene encoding the antibody light chain and a second gene encoding an antibody heavy chain must be introduced into the cell and expressed within an acceptable range of ratios. Expression of an unacceptable ratio of antibody light to heavy chain within the same cell or culture system may result in a highly inefficient production of the desired multimeric complex or in cell or organismal toxicity.
Past approaches for expressing polypeptide multimers in eukaryotic cells include introducing two or more vectors, each vector separately encoding each of the different polypeptides that make up the polypeptide multimer. Each vector typically carries a promoter driving expression of one polypeptide of the complex, and at least one vector typically encodes a selection marker. The vectors are then, in series or together, introduced into a cell (usually by transfection) and the cells are co-selected for the expression of both selection markers.
In another approach, a coding sequence along with a promoter for each polypeptide making up the polypeptide complex, is engineered into a single vector. This approach eliminates the need for working with multiple vectors, but still does not eliminate the potential for promoter competition between each coding sequence. Also, this approach can not typically resolve the problem of expressing the individual polypeptides comprising the protein multimer in an acceptable ratio to result in efficient expression of the protein multimer.
Thus, in either of the above approaches, a consistent ratio of the two products may not always be obtained in the host cell. This can be due to factors such as differential promoter activity, promoter competition for cellular factors required for optimal expression, efficiency of transcription and/or translation of the protein multimer component polypeptides, and/or a difference in the copy number for each of the vectors introduced into the cell.
Splicing vectors utitlizing a single splice donor and splice acceptor have also been developed. U.S. Pat. No. 5,043,270 discloses a minigene expressing a selectable marker, e.g. DHFR, and has an intron that contains a gene encoding a protein of interest. U.S. Pat. No. 5,561,053 discloses the inverse situation, in which the gene encoding a protein of interest contains an intron 5′ to the coding sequence. This intron contains a gene encoding a selectable marker bounded by the splice donor and acceptor. This type of intronic expression vector is further described in Lukas, B. K., et al. Nucleic Acids Res. 24:1774-1779 (1996). U.S. Pat. Appl. Pub. No. 2005/0019925 A1 discloses similar intronic vectors with a fusion selectable marker. It also discloses the use of two pairs of splice donors and splice acceptors for the expression of more than one protein of interest. All of these published constructs, however, rely on pairs of splice donors and splice acceptors, i.e., have one splice donor matched to a single splice acceptor. Each of these constructs depends on highly efficient splicing at all sites for effectiveness. There is no reference to using a single splice donor to activate alternative splicing from more than one splice acceptor to express multiple polypeptides. Further, there is no suggestion of the desirability of expressing the polypeptides at different ratios, or the substitution of different splicing acceptors to control the relative expression of the polypeptides.
Accordingly, a need exists for a construct that links the expression of two or more genes in a consistent ratio, such that the resultant gene products are efficiently produced and assembled.